Eucaryotic cells possess plasma membrane-associated transport proteins which actively efflux a variety of chemical compounds from the cells (Gottesman et al., Ann. Rev. Biochem. 62: 385 (1993)). The purpose of these transport proteins is to protect cells from cytotoxic and/or mutagenic compounds encountered in the diet or environment. However, these transport proteins are often very effective at removing pharmaceutical agents from target cells, thereby severely restricting their therapeutic efficacy. Consequently, compounds which inhibit these transport proteins are expected to enhance the clinical utility of drugs susceptible to such transport by enhancing their accumulation in target cells.
Two transport proteins which are important in the treatment of human diseases are termed P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP). Each of these proteins is highly effective in removing a variety of compounds from eucaryotic cells, using energy released by the hydrolysis of ATP. Because of their involvement in several human diseases, further discussed below, there is great interest in developing pharmaceutical agents which will effectively inhibit the abilities of these proteins to transport drugs. Additional transport proteins have been identified more recently, including cMOAT and additional proteins related to MRP.
An important issue regarding Pgp and MRP relates to their substrate specificity. Pharmacological comparison of cells overexpressing either Pgp or MRP have demonstrated that the resistance profiles conferred by these two transporters are only partially overlapping . For example, MRP-transfected cells demonstrate greater resistance factors for vincristine, etoposide and doxorubicin, than for vinblastine and paclitaxel (Cole et al., Cancer Res. 54: 5901 (1994)); whereas Pgp-overexpressing cells are extremely resistant to the later two drugs (Smith et al., Cancer 75: 2597 (1995)). This differential pharmacology indicates the feasibility of developing selective inhibitors of these transporters, which should provide methods useful for increasing the therapeutic efficacy of many types of pharmaceutical agents.
Another significant difference between Pgp and MRP relates to their distributions in normal tissues. P-glycoprotein has been shown to be expressed by several types of secretory cells, such as capillary endothelial cells in the brain and testis, and at sites within the pancreas, kidney and liver (Leveille-Webster and Arias, J. Membrane Biol. 143: 89 (1995)). In contrast, the expression of MRP mRNA occurs in virtually every type of tissue (Zaman et al., Cancer Res. 53: 1747 (1993)). Cells altered in disease states also differentially express Pgp and MRP, indicating that selective inhibitors will be preferred as therapeutic agents.
An example of transport protein-mediated drug resistance is the phenomenon of multidrug resistance (MDR) often encountered in cancer chemotherapy (Gottesman et al., Ann. Rev. Biochem. 62: 385 (1993)). In this situation, the proliferation of tumor cells that are resistant to many structurally unrelated drugs often results in the failure of chemotherapy. Tumor cells from patients undergoing chemotherapy often demonstrate elevated Pgp expression, suggesting that this mechanism of MDR is clinically important (Goldstein et al., J. Natl. Cancer Inst. USA 81: 116 (1989)). Recent studies have indicated that MRP is expressed in a high percentage of solid tumors and leukemias. However, no differences in MRP levels were detected between normal and malignant hematopoietic cells (Abbaszadegan et al., Cancer Res. 54: 4676 (1994)), and MRP levels were found to be lower in some tumors than in corresponding normal tissue (Thomas et al., Eur. J. Cancer 30A: 1705 (1994)). Therefore, it seems that different tumors will display different patterns of expression of Pgp, MRP and, possibly, other transporters.
Another example of drug transporter-mediated resistance is encountered in the effort to deliver drugs to the central nervous system, the testes and the eye. The blood-brain barrier exists to exclude toxic agents from the brain, and largely derives from the high level of expression of Pgp by endothelial cells in the capillaries of the brain (Schinkel et al., J. Clin. Invest. 97: 2517 (1996)). P-glycoprotein is similarly highly expressed in capillary endothelial cells in the eye (Holash and Stewart, Brain Res. 629: 218 (1993)) and testes (Holash et al., Proc. Natl. Acad. Sci. USA 90: 11069 (1993)), restricting the uptake of many compounds by these tissues. While these systems are useful in protecting normal tissues, they also impair the delivery of therapeutic agents to these sites when such delivery may be desired. For example, Pgp in brain capillary cells impairs effective treatment of brain tumors or neurological disease by drugs which are transported by this protein. Pgp is also highly expressed in the liver, adrenal, and kidney (Lum and Gosland, Hematol. Oncol. Clin. North Amer. 9: 319 (1995)), tissues in which drug delivery is restricted. It is envisioned that inhibition of Pgp or other transporters will facilitate drug delivery to these sites and so enhance the effectiveness of chemotherapy. It is also envisioned that antagonists of drug transporters will be useful in suppressing the secretion of endogenous compounds, including steroid hormones and cholesterol, providing therapeutic benefit in conditions in which excessive circulating levels of these compounds promote disease states.
Another example of drug transporter-mediated resistance is encountered in the effort to orally deliver therapeutic agents. P-glycoprotein is highly expressed at the brush-border membrane of the small intestine which reduces the bioavailability of orally administered drugs subject to transport (Sparreboom et al., Proc. Natl. Acad. Sci. USA 94: 2013 (1997)). It is envisioned that inhibition of Pgp or other transporters will facilitate drug absorption and so enhance the effectiveness of chemotherapy.
Another example of drug transporter-mediated resistance is encountered in the effort to deliver therapeutic agents to certain leukocytes. P-glycoprotein is highly expressed by certain subtypes of lymphocytes, natural killer cells and bone marrow stem cells (Gupta and Gollapudi, J. Clin. Immunol. 13: 289 (1993)). This reduces the therapeutic efficacy of drugs targeting these cells, including anti-HIV compounds for the treatment of AIDS (Yusa et al., Biochem. Biophvs. Res. Com. 169: 986 (1990)). Furthermore, the release of inflammatory cytokines and other immunomodulators appears to involve drug transporters (Salmon and Dalton, J. Rheumatol. 23 (suppl. 44): 97 (1996)). It is envisioned that inhibition of Pgp or other transporters will facilitate drug accumulation in these cells and so enhance the effectiveness of chemotherapy.
Organisms other than mammals also possess transport proteins similar to Pgp which have been shown to confer resistance to chemotherapeutic agents (Ullman, J. Bioenergetics Biomembranes 27: 77 (1995)). While the pharmacology of these transporters is not identical to that of Pgp, certain modulators are able to inhibit drug transport by both Pgp and protozoan transporters (Frappier et al., Antimicrob. Agents Chemother. 40: 1476 (1996)). It is envisioned that certain MDR modulators will facilitate drug accumulation in non-mammalian cells and so enhance the effectiveness of anti-infection chemotherapy.
The preceding discussion demonstrates that drug transporters are involved in determining the success of chemotherapy in a variety of disease states. While a variety of compounds have been shown to reverse transporter-mediated MDR in cell culture (i.e. act as MDR modulators), the clinical success with these agents has been unimpressive, predominantly due to the intrinsic toxicity of heretofore used modulators, and their undesired effects on the pharmacokinetics of the accompanying drugs. However, a likely cause of the failure of these agents is their lack of selectivity for different drug transporters. For example, inhibition of MRP by MDR modulators is likely to increase the uptake of cytotoxic anticancer drugs by many normal tissues, thereby producing greater untoward toxicity for the patient. Successful chemotherapy will consequently require a panel of transporter antagonists with differential selectivity for Pgp and MRP which will allow selection of the appropriate sensitizing agent.